Recreating functional vasculature is a pivotal step in the development of novel therapies in the field of regenerative medicine, providing innovative treatment options for patients suffering from vascular disorders, and generating functional and transplantable tissues that have been engineered in vitro. Endothelial cells (ECs), which comprise the inner lining of the vasculature, are critical cells to these endeavors. Because of their self-renewal capability and pluripotent nature, human pluripotent stem cells (hPSCs) can be harnessed to produce large populations of a desired cell type, including ECs. Human induced pluripotent stem cells (hiPSCs) are a recent class of hPSCs, which offer the possibility to advance regenerative medicine by providing patient-specific therapies. Controlled and robust differentiation of hiPSCs toward vascular lineages is critical for the advancement and future of patient-specific vascular therapeutics. The goal of the proposed research is to investigate EC differentiation from hiPSCs, and their specification into arterial and venous fate, with a long term goal. Our aims are to: (1) generate a pure EC population from fibroblast-derived hiPSCs in a 2D, feeder-free culture; (2) compare EC differentiation from hiPSCs derived from various origin cell types and derivation techniques; and (3) induce specification of arterial and venous ECs from a common progenitor population. To achieve these aims, the proposed research strategy involves a unique combination of methods in stem cell and vascular biology engineering. Successful completion of these aims has considerable clinical impact with respect to improved vascular therapeutics and could broaden our understanding of vascular development and repair. PUBLIC HEALTH RELEVANCE: Recreating functional vasculature is a pivotal step in the development of novel therapies in the field of regenerative medicine, providing innovative treatment options for patients suffering from vascular disorders, and generating functional and transplantable tissues that have been engineered in vitro. Because of their self- renewal capability and pluripotent nature, human induced pluripotent stem cells (hiPSCs) can be harnessed to produce large populations of a desired cell type, including endothelial cells (ECs), which form the inner lining of the vasculature. This proposal aims to examine the potential of hiPSCs to differentiate into a homogenous population of mature and functional ECs, to understand whether hiPSC origin type affects endothelial potential, and to induce arterial and venous specification via biochemical and biophysical means.